The present invention relates to a novel human gamma-butyrobetaine hydroxylase (xcex3-BBH). The invention also relates to polynucleotides encoding the xcex3-BBH polypeptides. The invention further relates to methods using the polypeptides and polynucleotides as targets for the diagnosis and treatment related to xcex3-BBH mediated or -related disorders. The invention further relates to drug-screening methods using the xcex3-BBH polypeptides and polynucleotides to identify agonists and antagonists for diagnosis and treatment. The invention further encompasses agonists and antagonists based on the xcex3-BBH polypeptides and polynucleotides. The invention further relates to procedures for producing the xcex3-BBH polypeptides and polynucleotides.
Carnitine (3-hydroxy-4-N-trimethylaminobutyrate) biosynthesis is essential for the xcex2-oxidation of fatty acids in eukaryotic mitochondria. Carnitine plays an essential role in the transport of activated fatty acids across the mitochondrial membrane (Lehninger et al. (1993) Principles of Biochemistry, 2d Edition). Many organisms, from bacteria to humans, are able to synthesize carnitine (Vaz F.M. (1998) Biochemical and Biophysical Res. Comm. 250: 506-510). The concentration of carnitine in different species and different tissues varies over a wide range. In mammalian tissues, the concentration varies between 0.1 and a few millimoles per liter (Bremer, J. (1983) Physiological Reviews, Vol. 63, No.4, p.1420-1480). Carnitine is synthesized from the amino acids lysine and methionine. There are several steps (5 in total) involved in the synthesis of carnitine. The last step in the carnitine biosynthetic pathway requires the enzyme xcex3-BBH. It catalyzes the reaction of hydroxylation of gamma-butyrobetaine to carnitine. In humans, this final reaction occurs in liver, kidney, and brain tissue but not in cardiac or skeletal muscle (Engel, A. G. and C. J. Rebouche (1984) J Inher. Metab. Dis. 7 Suppl., 38-43).
The xcex3-BBH belongs to a unique class of non-heme ferrous iron dioxygenases in which the hydroxylation of susbstrate is linked to the oxidative decarboxylation of xcex1-ketoglutarate (Abbott, M. and S. Udenfriend (1974) in Molecular Mechanisms of Oxygen Activation (Hayaishi,O. ed.) pp. 167-214, Academic, Orlando, Fla.). xcex3-BBH requires xcex1-ketoglutarate, Fe+2 and molecular oxygen as cofactors. Of all the enzymes in the carnitine biosynthetic pathway, xcex3-BBH is the best-studied enzyme (Vaz, F. M. (1998) Biochemical and Biophysical Res. Comm. 250:506-510).
The mechanism of fatty acid transport across the mitochondrial membrane involves the activation and transport of the fatty acids across the membrane. The free fatty acids that enter the cytosol from the host bloodstream cannot pass directly through the membranes, but must first undergo a series of enzymatic reactions. The first is characterized by a family of isozymes present in the outer mitochondrial membrane which includes the acyl-CoA-synthetases. The different synthetase isozymes act on the fatty acids of short, intermediate, and long chain length. The acyl-CoA-synthetases catalyze the formation of a thioester linkage between the fatty acid carboxyl group and the thiol group of the coenzyme A to yield a fatty-acyl-CoA. The fatty acyl-CoA molecules are high energy compounds.
Fatty acyl-CoA esters formed in the outer mitochondrial membrane do not cross the inner mitochondrial membrane intact. Instead, the fatty acyl group is transiently attached to the hydroxyl group of carnitine. It is the fatty acyl-carnitine that is carried across the inner mitochondrial membrane by a specific transporter (Lehninger et al. (1993) Principles of Biochemistry, 2d Edition). The second step in transport involves the enzyme carnitine acyltransferase I which catalyzes the trans-esterification of the fatty acyl group from coenzyme A to carnitine. The fatty-acyl carnitine ester crosses the inner mitochondrial membrane into the matrix by facilitated diffusion through the acyl-carnitine/carnitine transporter. The third and final step of the entry process involves the enzymatic transfer of the fatty acyl group from carnitine to intramitochondrial coenzyme A by carnitine acyltransferase II.
Until recently, there was little molecular characterization of the enzymes involved in carnitine biosynethesis (Vaz, F. M. (1998) Biochemical and Biophysical Res. Comm. 250:506-510). Since xcex3-BBH is the last enzyme in the biosynthesis of carnitine it is a major target for drug action and development.
Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown butyrobetaine hydroxylases. The present invention advances the state of the art by providing a previously unidentified human xcex3-BBH.
It is an object of the invention to identify novel xcex3-BBHs (2-oxoglutarate dioxygenases).
It is a further object of the invention to provide novel xcex3-BBHs that are useful as reagents or targets in xcex3-BBH assays applicable to treatment and diagnosis of human xcex3-BBH disorders as relates to aberrant carnitine biosynthesis.
It is a further object of the invention to provide polynucleotides corresponding to the novel xcex3-BBH polypeptides that are useful as targets and reagents in xcex3-BBH assays applicable to treatment and diagnosis of xcex3-BBH-related disorders and useful for producing novel xcex3-BBH polypeptides by recombinant methods.
A specific object of the invention is to identify compounds that act as agonists and antagonists that can modulate the expression of the novel xcex3-BBH.
A further specific object of the invention is to provide compounds that modulate expression of the xcex3-BBH for treatment and diagnosis of xcex3-BBH related disorders.
The invention is thus based on the identification of a novel human xcex3-BBH. The amino acid sequence of the xcex3-BBH is shown in SEQ ID NO 1. The nucleotide sequence is shown in SEQ ID NO 2.
The invention also provides variant polypetides having an amino acid sequence that is substantially homologous to the amino acid sequence shown in SEQ ID NO 1.
The invention also provides variant nucleic acid sequences that are substantially homologous to the nucleotide sequences shown in SEQ ID NO 2.
The invention further provides nucleic acid constructs comprising the nucleic acid molecules described herein. In a preferred embodiment, the nucleic acid molecules of the invention are operatively linked to a regulatory sequence.
The invention also provides vectors and host cells for expressing the xcex3-BBH nucleic acid molecules and polypeptides, and particularly recombinant vectors and host cells.
The invention also provides methods of making the vectors and host cells and methods for using them to produce the xcex3-BBH nucleic acid molecules and polypeptides.
The invention also provides antibodies or antigen-binding fragments thereof that selectively bind the xcex3-BBH polypeptides and fragments.
The invention also provides methods of screening for compounds that modulate expression or activity of the xcex3-BBH polypeptides or nucleic acid (RNA or DNA).
The invention also provides a process for modulating xcex3-BBH polypeptide or nucleic acid expression or activity, especially using the screened compounds. Modulation may be used to treat conditions related to aberrant activity or expression of the xcex3-BBH polypeptides or nucleic acids.
The invention also provides assays for determining the activity of or the presence or absence of the xcex3-BBH polypeptides or nucleic acid molecules in a biological sample, including for disease diagnosis.
The invention also provides assays for determining the presence of a mutation in the polypeptides or nucleic acid molecules, including for disease diagnosis.
In still a further embodiment, the invention provides a computer readable means containing the nucleotide and/or amino acid sequences of the nucleic acids and polypeptides of the invention, respectively.